Pharmacokinetics and Adverse Events Following 5-Day Repeated

Home , Lenograstim

Bone Marrow Transplantation (2000) 26, 939–946  2000 Macmillan Publishers Ltd All rights reserved 0268–3369/00 $15.00 www.nature.com/bmt Pharmacokinetics and adverse events following 5-day repeated administration of lenograstim, a recombinant human granulocyte colony-stimulating factor, in healthy subjects

S Akizuki1, F Mizorogi2, T Inoue1,2, K Sudo1 and A Ohnishi1,2

1Departments of Laboratory Medicine and 2Internal Medicine, Daisan Hospital, Jikei University School of Medicine, Komae, Tokyo, Japan

Summary: ipheral blood progenitor cell; allogeneic transplantation; thrombocytopenia; pharmacokinetics Recombinant human granulocyte colony-stimulating factor (rhG-CSF) (lenograstim) was administered to healthy subjects at doses of 2, 5 and 10 ␮g/kg/day for 5 days (twice a day subcutaneously) to examine the opti- Recombinant human granulocyte colony-stimulating factor mal dose and schedule of lenograstim in mobilizing peri- (rhG-CSF) has been widely used to treat patients with neu- pheral blood progenitor cells (PBSC) for allogeneic tropenia during intensive chemotherapy.1–3 Recently, rhG- transplantation. Lenograstim administration signifi- CSF has been applied to peripheral blood progenitor cell cantly increased CD34+ cells in a dose-related manner. (PBSC) allogeneic transplantation from healthy donors, A significant correlation was observed between the because administration of rhG-CSF in healthy donors mobi- maximal post-dosing counts and the pre-dosing baseline lizes a sufficient number of PBSC that can be harvested.4–11 counts of CD34+ cells. Peripheral neutrophils increased Several preliminary studies on PBSC transplantation have markedly by seven to 13 times from the baseline to a been reported and the appropriate dose and schedule of peak of approximately 40 000/␮l on day 5 for the 5 and rhG-CSF treatment in healthy donors have been dis- 10 ␮g/kg/day doses. After peak serum concentration cussed.4–11 The definitive method, however, remains controversial.6,9,12–14 In addition, repeated dosing of rhG-CSF (Cmax) was attained 4 h following administration, serum G-CSF declined with time in a log-linear fashion. The in healthy subjects has been reported to cause a variety of adverse events including bone pain, low grade fever, Cmax and 12 h area-under-the-curve increased dose dependently, but minimum drug level increased up to increases in transaminases, lactate dehydrogenase (LDH) day 2 and then decreased until day 5. Clearance and alkaline phosphatase (ALP) and platelet decreased with increasing dosage at the first dose, and reduction.8,10,14–16 Therefore the optimized dose and dur- increased significantly at the last dose. We found a ation are determined by the balance between the dose- highly significant correlation between absolute neutro- related adverse events and the required harvest of PBSC phil counts and clearance for each dose. Adverse events from healthy donors. The primary aim of this study was to most frequently occurred on day 6, with increases of evaluate the relationship between the dose and mobilized alkaline phosphatase and lactate dehydrogenase and PBSC count and between dose-related adverse events and onset of bone pain. Increases of aspartate amino- the time after dosing. transferase and alanine aminotransferase occurred as Pharmacokinetic data for G-CSF are required to resolve delayed events. Platelet count gradually decreased after the above issues. The kinetic disposition of G-CSF in neu- the end of drug administration to 57% of the pre-dosing tropenic patients receiving chemotherapy is well estab- count on day 10, but was still within the normal range. lished,17–20 but the dose-related kinetic profile after repeated These preliminary results suggest that repeated doses administration in healthy subjects is relatively unexplored, of lenograstim induce mobilization of PBSC in a dose- particularly that concerning drug clearance. G-CSF has dependent manner and the pre-dosing baseline count of been reported to be cleared by a non-saturable mechanism PBSC may predict the post-dosing maximal mobiliz- in the kidney/spleen and by a saturable mechanism in per- ation. The drug treatment may cause delayed-onset ipheral neutrophils and progenitor cells in bone marrow.21–23 moderate thrombocytopenia and increased transamin- Therefore our secondary aim was to evaluate the pharmacoki- ase, and the drug clearance changes in a complex man- netics of G-CSF in the 5 day repeated administration study. ner during repeated dosing. Bone Marrow Transplan- tation (2000) 26, 939–946. Keywords: granulocyte colony-stimulating factor; per- Materials and methods

Correspondence: Dr A Ohnishi, Department of Laboratory Medicine, Dai- Subjects san Hospital, Jikei University School of Medicine, 4–11–1 Izumihoncyo, Komae, Tokyo 201–8601, Japan A total of 18 healthy male Japanese volunteers 21 to 27 Received 1 March 2000; accepted 17 July 2000 years of age (mean: 23.6 years) and weighing between 51.0 Pharmacokinetics and Adverse Events of G-CSF S Akizuki et al 940 and 69.5 kg (mean ± s.d.: 59.7 ± 5.3) participated in the Measurement of serum G-CSF levels study after giving their informed consent. Each subject was judged to be healthy on the basis of a complete physical Serum samples were stored in small aliquots at −20°C until examination, a 12-lead electrocardiogram, hemogram, analysis. Serum G-CSF levels were measured by enzyme- coagulation profile, blood chemistry profile, and urinalysis. linked immunosorbent assay (ELISA).25 All subjects were normotensive and had a normal heart rate. Due to the anticipation of bone pain and fever, the volunteers were offered the possibility of taking nonsteroidal Pharmacokinetic analysis anti-inflammatory drugs (NSAID) when giving their informed consent. Ultimately, none of the volunteers received Serum G-CSF concentrations were fitted to a pharmaco- NSAID or any other medication apart from rhG-CSF kinetic model by the nonlinear least squares method, using throughout the study. The subjects entered the Research the computer program Window-Nonlin (Version 4.0, SCI Ward in Kannondai Clinic (Ibaraki, Japan) at least 24 h Software, Pharsight, Lexington, KY, USA).26 Peak serum before the study began, and stayed throughout the study concentrations (Cmax) and time to reach Cmax (tmax) were period. The study protocols were approved by the obtained from real data points. The area-under-the-curve Institutional Ethical Committee. (AUC) after a single dose was calculated using log-linear trapezoidal approximation from time 0 to the time of the last measurement in the concentration – time curve and G-CSF administration and blood sampling AUC0–ϱ was calculated by summing the AUCs obtained Lenograstim (recombinant human G-CSF; Chugai Pharma- from 0 to the last measured serum concentration divided ceutical, Tokyo, Japan) was administered subcutaneously by elimination constant (K). The subcutaneous clearance for 5 consecutive days (days 1 to 5) at a dose of 2, 5 or (CL/F0–12 h) on day 1 was estimated from the s.c. 10 ␮g/kg/day in six volunteers for each rhG-CSF dose dose/AUC0–ϱ, and the CL/F96 to 108 h on day 5 was estimated level. Each daily dose was divided into two injections to from the s.c. dose/AUC96 to 108 h, where F is the subcutane- reduce subjective symptoms (1, 2.5 or 5 ␮g/kg/twice/day, ous bioavailability that could not be determined. The mini- every 12 h). mum serum concentration of G-CSF (Cmin) was obtained Peripheral blood samples were collected every morning from the real data point just before the next morning dose. (before drug administration on days 1 to 5) for determining Dose/Cmin was used in this study as a parallel and accept- white blood cell (WBC) counts, neutrophil counts, and able parameter to the CL/F. blood biochemistry including alkaline phosphatase (ALP), lactate dehydrogenase (LDH), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) from day 1 Safety assessment (before the 1st dose) to day 10, and then on day 17 (follow- up). To determine the absolute number of PBSC, hep- The subjects stayed in the research unit from day 1 to day arinized peripheral blood samples were collected on days 10. Safety evaluation of the repeated doses of rhG-CSF was 1 (before the 1st dose), 2, 3, 4, 5, 6, 8 and 10. Serum G- based on assessments of the results of physical examin- CSF was measured before each lenograstim injection from ations, subjective symptoms, and vital signs including day 1 to day 5, and serially from 0 to 12 h (day 1) and blood pressure and heart rates throughout the study period. from 96 to 168 h (day 5). Blood samples for CD34+ cell All side-effects (adverse events) were noted on the appro- assays were taken before dosing in the mornings of days 1 priate case report forms with a brief description specifying (before the 1st dose), 2, 3, 4, 5, 6, 8 and 10. Blood cell the nature, severity, date, time of occurrence, duration, and components (lymphocyte, monocyte, eosinophil, basophil, the investigator’s opinion of whether the reaction was test platelet, myelocyte, metamyelocyte and erythrocyte) were compound-related or incidental. evaluated from day 1 (before the 1st dose) to 10 and on day 17. WBC were measured using an automated cell counter (Sysmex NE-8000: TOA, Kobe, Japan). The cell compo- Statistical methods nents were estimated after manual leukocyte differential. Data are expressed as mean ± standard error of the mean Flow cytometric analysis of CD34+ cells and their (s.e.m.) unless otherwise indicated. Multi-group compari- subsets sons were performed by analysis of variance (ANOVA). Spearman’s rank correlation coefficient (R) was calculated The peripheral blood samples were also analyzed by dou- to estimate correlations. All the statistical analyses were 24 ble-color flow cytometry. After lysis with NH4Cl, the cell performed using the Stat View program. Correlations concentration was adjusted to 1 × 106 cells/ml in PBS with between the pre-treatment baseline counts of CD34+ cells 10% mouse serum. The cells were stained simultaneously and the post-treatment maximal mobilized counts of CD34+ with phycoerythrin (PE)-conjugated anti-CD34 antibody cells for three doses of lenograstim were analyzed by mul- (Immu133) for 30 min at 4°C in the dark. As isotype con- tiple regression analysis (SAS release 6.12 procedure Reg). trols for the staining, we used PE-conjugated mouse IgG1. A regression equation was obtained that expressed the Flow cytometric analysis was performed with a FACScan relationship between the predicted and the observed (Becton Dickinson, Tokyo, Japan). The cells were selected maximal mobilized count of CD34+ cells. The minimum for their small size and the expression of the CD34 antigen. statistically significant level was set at P Ͻ 0.05.

Bone Marrow Transplantation Pharmacokinetics and Adverse Events of G-CSF S Akizuki et al 941 Results Table 1 Changes in clinical laboratory test values during repeated- dose study period Effect of lenograstim treatment on WBC counts, + Clinical test Unit Dose Peak value tmax (term) Fold neutrophil counts and on PBSC mobilization as CD34 ␮ cells ( g/kg/day) Signiﬁcant increases in WBC and neutrophil counts were 2 25.3 ± 3.3 5 (2–7) 4.4 observed in all volunteers administered 5 and 10 ␮g/kg/day WBC (×109/l) 5 51.0 ± 16.7 5 (2–7) 9.4 ␮ 10 51.2 ± 8.7 5 (2–7) 9.2 compared to 2 g/kg/day. Peripheral neutrophils increased 2 20.7 ± 2.5 5 (2–7) 6.8 markedly by about seven to 13-fold from the baseline Neutrophil (×109/l) 5 41.0 ± 13.2 5 (2–7) 12.6 count, and the peak count of approximately 40 × 109/l with 10 38.9 ± 7.0 5 (2–7) 10.5 the two upper doses was reached on day 5. The increments 2 19.2 ± 13.6 6 (5–6) 13.6 + ␮ ± were similar for 5 and 10 ␮g/kg/day (Figure 1b and CD34 cell (/ l) 5 62.5 41.4 5 (4–6) 36.8 10 94.7 ± 50.5 5 (4–6) 44.1 Table 1). PBSC counts in peripheral blood measured as ± + 2 282 20 6 (4–10) 1.7 CD34 cells increased dose-dependently to a peak count on ALP (IU/l) 5 379 ± 124 6 (4–10) 2.2 day 5 to 6, and the increase was approximately 14- to 44- 10 404 ± 44 6 (4–10) 2.6 fold compared with the corresponding predose values as 2 336 ± 63 6 (4–6) 1.4 LDH (IU/l) 5 553 ± 157 6 (4–8) 2.2 shown in Table 1 and Figure 1a. 10 815 ± 178 6 (4–8) 3.2 There was a signiﬁcant correlation between the predose 220± 810Ͻ 1.5 ALT (IU/l) 5 42 ± 25 10Ͻ 3.6 10 71 ± 90 10Ͻ 4.9 ± Ͻ 120 a 217310 1.1 AST (IU/l) 5 32 ± 14 10Ͻ 2.0 10 53 ± 61 10Ͻ 4.1 100

l) All data indicate mean ± s.e.m. m /

4 80 WBC = white blood cell; ALP = alkaline phosphatase; LDH = lactase dehydrogenase; ALT = alanine aminotransferase; AST = asparate amino- ×10 transferase. 60 The peak value was obtained as observed data during the study period

and time to reach the peak value was deﬁned as tmax (term) is deﬁned as cells ( + the period with values exceeding the normal ranges. The fold was obtained 40 by comparing the predose value. CD34 20 baseline counts of CD34+ cells and the post-dose maximal mobilized counts of CD34+ cells for all three doses of leno- 0 grastim as determined by analysis of variance. Figure 2a 1 2 3 4 5 6 7 8 9 10 demonstrates the relationship between predose CD34+ cell counts (x-axis), maximally mobilized post-dose CD34+ cell counts (y-axis) and rhG-CSF dose (z-axis). It indicates that 45 b higher doses of rhG-CSF and greater predose CD34+ cell + 40 counts are associated with mobilization of more CD34 cells. A multiple regression analysis of the relationship 35 among the predose CD34+ cell count, the maximally mobil-

/l) + 9 30 ized post-dose CD34 cell count, and the rhG-CSF dose was performed. A highly signiﬁcant correlation was ×10 25 observed between the observed and predicted value of the + 20 maximal post-dose mobilization of CD34 cells by multiple regression analysis. The regression equation was: predicted 15 maximal CD34+ cell counts = 13.279 × dose + 28.51 × the Neutrophil ( 10 predose count − 25.16 (contribution ratio: 65.1%) (Figure 2b). 5

0 Serum G-CSF concentration-time relationship and 1102 3 4 5 6 7 8 9 pharmacokinetic parameters

Lenograstim After the peak serum concentration (Cmax) was attained 4 h Time (days) following administration, serum G-CSF declined with time in a log-linear fashion, and the decline tended to be less + Figure 1 Changes in absolute CD34 cell count and absolute neutrophil steep with increasing rhG-CSF dosage. The concentration- count. Changes in absolute CD34+ cell count (a), and absolute neutrophil count (b) with time during and after the repeated-dose administration of time curves varied dose-dependently as shown in Figure 3a. rhG-CSF (lenograstim) at doses of 2 (᭿), 5 (᭹) and 10 (̆) ␮g/kg/day On day 1 after the ﬁrst dose, Cmax and the 12 h AUC (twice a day for 5 days). increased dose dependently (Table 2). However, the mini-

Bone Marrow Transplantation Pharmacokinetics and Adverse Events of G-CSF S Akizuki et al 942

l) 100000

m a a

160 10000 140

120 cell counts (/ counts cell

+ 100 1000 80 60 100 40 7 20

6 G-CSF (pg/ml) (day 1) 0 5 10 4 g/kg) 4 m 3.5 3 Pre dose3 CD34 2.5 2 1

Observed maximum CD34 maximum Observed 2 0 2 4 6 8 12 1.5 1 + cells counts1 (/ rh G-CSF ( 0.5 0 0 Lenograstim m l) Time (h) on day 1

100000 b l) m 160 b 10000 ) 140 min 1000 120 cell counts (/ + 100 100 80 G-CSF (pg/ml) (C 60 10

40 1 20 1 2 3 4 5 678 Adj R2 = 0.6514 0

Observed maximum CD34 Lenograstim 0 20 40 60 80 100 120 140 160 Time (days) of study Predicted maximum CD34+ cell counts (/m l) Figure 3 Serum G-CSF concentration–time relationship. (a) Serum G- 13.279278 × dose + 28.506731 × predose value 25.161428 CSF concentration–time relationships after the ﬁrst dose of rhG-CSF

(lenograstim) at 1 (᭿), 2.5 (᭹) and 5 (̆) ␮g/kg. (b)Cmin–time relation- Figure 2 Relationship between the observed and predicted value of the ships during and after repeated-dose administration of rhG-CSF maximal post-dose mobilization of CD34+ cell. (a) Relationship among + (lenograstim) at doses of 2 (᭿), 5 (᭹) and 10 (̆) ␮g/kg/day (twice a day predose CD34 cell counts (x-axis), maximally mobilized post-dose for 5 days). C = minimum drug level just before the next morning dose. CD34+ cell counts (y-axis) and dose (z-axis) indicates that higher doses min of rhG-CSF and greater counts of the predose CD34+ cell induce mobiliz- + ation of more CD34 cell. (b) Multiple regression analysis demonstrates ± ± ± highly significant correlation between the observed and predicted value of 104.2 20.6 ml/h/kg; dose/Cmin: 8.3 1.0 vs 4.5 0.7 and the maximal post-dose mobilization of CD34+ cell (the predicted maximal 5.1 ± 1.5 l/kg, respectively) (Table 2). Changes in the above CD34+ cell counts = 13.279 × dose + 28.51 × predose cell count − 25.16) two parameters (CL/F, dose/Cmin) with time were almost (contribution ratio: 65.1%). The symbols indicate the value in the rhG-CSF consistent on days 1 and 5. dosing of 2 (᭿), 5 (᭹) and 10 (̆) ␮g/kg/day (twice a day for 5h days). We found a highly significant correlation between absolute neutrophil counts and dose/Cmin for each dose mum drug level (Cmin) (just before the next morning dose) (Figure 4). increased up to day 2, then decreased gradually until day 5 despite the repeated doses. The decreases were especially Adverse events including laboratory test abnormalities marked at the doses of 5 and 10 ␮g/kg/day (Figure 3b). ␮ Both clearance (CL/F) and dose/Cmin decreased with All subjects who received 5 and 10 g/kg/day complained increasing dosage on day 1 (Table 2). Both parameters of mild to moderate bone pain (peak on day 5; duration of increased significantly at the last dose (day 5) of the 5-day 3 to 5 days). Transient low grade fever (duration: 1–3 days) repeated administration, but the magnitudes were signifi- was observed in 10 of 18 subjects (two of six for 2 ␮g, cantly higher at the dose of 5 ␮g/kg/day compared to 2 four of six for 5 ␮g, and four of six for 10 ␮g/kg/day). Mild and 10 ␮g/kg/day (CL/F: 199.8 ± 17.7 vs 117.9 ± 9.7 and headache was complained of by three of six subjects on

Bone Marrow Transplantation Pharmacokinetics and Adverse Events of G-CSF S Akizuki et al 943 Table 2 Pharmacokinetic parameters of lenograstim on day 1 and day 5 in repeated-dose administration

Parameters Unit Day 1 (0–12 h) dose (␮g/kg/day)a Day 5 (96–108 h) dose (␮g/kg/day)

251025 10

AUCb (␮g.h/l) 12.55 ± 1.05 40.92 ± 3.93d 239.08 ± 52.04d 7.31 ± 0.43f 11.61 ± 0.98d,f 48.62 ± 8.94d,f CL/F (ml/h/kg) 82.4 ± 6.6 63.9 ± 6.0d 26.4 ± 5.3d 117.9 ± 9.7e 199.8 ± 17.7d,f 104.2 ± 20.6e c ± ± ± d ± ± d,f ± f Dose/Cmin ) (l/kg) 4.20 0.46 2.59 0.25 0.85 0.21 4.51 0.65 8.27 1.04 5.05 1.45

All data indicate mean ± s.e.m. aDose was given divided into two injections: 1, 2.5, 5 ␮g/kg/twice/day, every 12 h. b On day 1 AUC was calculated as AUC0–ϱ, while that on day 5 was calculated as AUC96–108 h. CL/F, subcutaneous clearance is calculated by Dose/AUC. c Dose/Cmin on day 1 was by dividing the dose obtained by the serum level just before the day 2 morning dose, while that on day 5 was obtained by dividing the dose by the serum level 24 h after the last dose. dP Ͼ 0.01 compared with 2 ␮g/kg/day; eP Ͻ 0.05, fP Ͻ 0.01 compared with day 1.

2 m g/kg/day 5 m g/kg/day 10 m g/kg/day 14 r = 0.260 14 r = 0.628 14 r = 0.723 slope = 0.0001589 slope = 0.0001491 slope = 0.0001498 12 12 12

10 10 10

8 8 8

(l/kg) 6 6 6 min

D/C 4 4 4

2 2 2

0 0 0 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 Neutrophil (×104/m l)

Figure 4 Correlations between absolute neutrophil counts and dose/Cmin. Correlations between absolute neutrophil counts and dose/Cmin in three lenograstim doses of 2 (᭿), 5 (᭹) and 10 (̆) ␮g/kg/day during the study period.

5 ␮g/kg/day and four of six subjects on 10 ␮g/kg/day, and using the formula reported by Ho¨glund et al6 the target of fatigue by two of six subjects on 5 ␮g/kg/day and four of mobilization is over 50 cells/␮l. The target PBSC counts six subjects on 10 ␮g/kg/day during the treatment period were attained in four of six subjects receiving 5 ␮g/kg/day (Figure 5). These adverse effects were tolerated without any and in five of six subjects receiving 10 ␮g/kg/day, which medication and no other undesirable clinical signs or symp- agreed with the previous study.6 toms were recognized during the study period. The platelet In the present study we adopted a twice-daily adminis- count remained unchanged during the treatment period, but tration schedule to reduce the inter-subject variability of gradually decreased after treatment was completed to 72 to mobilization as well as to avoid the peak serum G-CSF 57% of the predose value on days 7 to 10, and recovered level related side-effects. Furthermore, in clinical settings, to the predose value on day 17 (Figure 6). Dose-dependent we can discontinue the drug more easily with the twice- increases in ALP and LDH were observed, with levels daily regimen. A twice-daily dose schedule has been com- above the normal ranges on days 4 to 10 and peak levels pared with a single-daily dose schedule by some investi- on day 6 (peak day). Similarly, dose-dependent increases gators, who have reported better and shorter mobilization in transaminases (AST, ALT) were observed and the peak with a twice-daily schedule.9,28 However, the peak mobiliz- was on day 10 (Table 1). ation day was day 5 in our study which was similar to that observed in many single-daily dose studies. Furthermore better or shorter mobilization was also not observed in sev- Discussion eral other studies using divided-dose schedule. Eventually the mobilization results appear similar with either twice- The primary aim of using rhG-CSF in PBSC transplantation daily or single-daily administration. The issue as to which is to obtain sufficient mobilized PBSC (over 50/␮l for schedule is more efficient or safe for mobilization remains CD34+ cells) in healthy subjects at a rhG-CSF dose as low controversial. In our study, doses of 5 and 10 ␮g/kg/day as possible. This target count is based on the following were able to mobilize PBSC on day 5 but there was still calculation. The target PBSC count is generally agreed to inter-subject variability. We therefore attempted to analyze be over 5 × 106 CD34+ cells/kg recipient body weight.27 the relationships between the predose CD34+ cell count, the Assuming an average recipient body weight of 50 kg, and maximally mobilized post-dose CD34+ cell count, and rhG-

Bone Marrow Transplantation Pharmacokinetics and Adverse Events of G-CSF S Akizuki et al

944 Lenograstim Time (days)

1 2 3 4 5 6 7 8 91011121314151617

Pharmacological effects

WBC increase

Neutrophil increase

CD34+ cell increase

Adverse events Fever

Bone pain

ALP increase

LDH increase

ALT increase

AST increase

Platelet reduction

Figure 5 Adverse events after the rhG-CSF administration. Pharmacological effects, and adverse events including abnormal clinical laboratory test results during and after repeated-dose administration of rhG-CSF (lenograstim).

300 rhG-CSF administration in healthy subjects may be useful in PBSC transplantation. 250 In the present repeated dose study, in contrast to general

conceptions, Cmin decreased gradually from day 2 despite 200 repeated twice-a-day dosing, suggesting that G-CSF clear- /l) 9 ance may increase following repeated doses. Increased clearance following repeated doses has been reported by ×10 150 many investigators,18–20,29 but the day-by-day increase in drug clearance has not been demonstrated in healthy sub- 100

Platelet ( jects. Saturable and non-saturable processes are involved in the elimination of G-CSF.21–23 The G-CSF receptor on 50 neutrophils and progenitor cells in bone marrow is thought : normal range to be one of the saturable processes, while the kidney plays

0 a major role in non-saturable elimination of G-CSF. As 18–20,29 1234567891011 17 already reported elsewhere, we also demonstrated a signiﬁcant correlation between the increase in neutrophil

Lenograstim administration count and the dose/Cmin value, a parameter thought to corre- Time (days) spond to drug clearance, for all three doses. Surprisingly, the slopes of correlations for the three doses were almost Figure 6 Platelet count–time relationship during and after the rhG-CSF the same, ranging from 0.0001491 to 0.0001589. The ident- administration. Absolute platelet count–time relationships during and after ical slopes for the three doses suggest that increase in neu- repeated-dose administration of rhG-CSF (lenograstim) at doses of 2 (᭿), 5(᭹) and 10 (̆) ␮g/kg/day (twice a day for 5 days). The shadow area trophils by rhG-CSF administration enhances the saturable indicates the normal range of absolute platelet count. drug clearance (depends on binding to neutrophils) at the same rate for all three doses. However, because the neutrophil counts increase with rhG-CSF dose, the absolute clear- CSF dose. Then, we found a fairly significant correlation ance through the saturable process increases with increasing between the observed and predicted maximally mobilized rhG-CSF dose. Conversely, the relative contribution of post-dose CD34+ cell counts, suggesting that we may pre- unsaturable drug clearance (elimination from kidney, rela- dict the mobilized PBSC counts before the drug treatment. tively constant) to total clearance decreases with increased Subjects with a low peripheral PBSC count at baseline may dosage. This would explain the decrease in the y-intercept not respond sufficiently to repeated doses of rhG-CSF in with increasing doses. On the other hand, the findings from mobilizing PBSC. The prediction of mobilized PBSC by dose/Cmin correspond with the results of CL/F on days 1

Bone Marrow Transplantation Pharmacokinetics and Adverse Events of G-CSF S Akizuki et al 945 and 5. On day 1, CL/F decreased with increasing dosage, Acknowledgements however subsequent repeated doses increased CL/F significantly on day 5 compared with day 1. As shown in The authors thank Dr Shigetaka Asano, Department of Table 1, it is uncertain why CL/F was greater at the dose Hematology/Oncology, The Institute of Medica Science, Univer- of 5 ␮g/kg/day than at 10 ␮g/kg/day. One possible expla- sity of Tokyo for his supervision of this study. We also thank Dr nation of this inversion is that increases in neutrophils by Masayuki Kobayashi, Department of Hematology/Oncology, Jikei University School of Medicine and the staff of Clinical Research these two doses are of the same magnitude and saturated and Development Division, Chugai Pharmaceutical Co, Ltd, elimination by neutrophil and/or progenitor cells in bone Tokyo, Japan for their support. The authors also thank the mem- marrow probably already reaches the maximum between bers of Kannondai Clinic, Tsukuba, Japan for their help. the doses of 5 and 10 ␮g/kg/day. Consequently, the surplus drug reduces CL/F at the dose of 10 ␮g/kg/day. Whatever the reason(s), this change in the drug clearance should be References considered when choosing the appropriate dosage for PBSC transplantation. 1 Yoshida Y, Hirashima K, Asano S et al. A phase II trial of Concerning the adverse events during the repeated-dose recombinant human granulocyte colony-stimulating factor in study, we observed moderate platelet reduction in a dose- the myelodysplastic syndromes. Br J Haematol 1991; 78: 378–384. dependent manner. The platelet counts, despite no plas- 2 Ohno R, Tomonaga M, Ohshima T et al. A randomized con- mapheresis, decreased gradually day-by-day after the ter- trolled study of granulocyte colony-stimulating factor after mination of rhG-CSF treatment and were lowered maxi- intensive induction and consolidation therapy in patients with mally on day 10 to 57% of the pre-dose value. However, acute lymphoblastic leukemia (JACSG). Int J Hematol 1993; the counts were still within the normal range. A number of 58: 73–81. reports have demonstrated thrombocytopenia related to 3 Kotake T, Miki T, Akaza H et al. Effect of recombinant gra- rhG-CSF treatment.10,14,16 However, thrombocytopenia in nulocyte colony-stimulating factor (rhG-CSF) on chemo- these reports was caused either by apheresis or chemotherapy-induced neutropenia in patients with urogenital can- therapy. Delayed-onset thrombocytopenia in healthy sub- cer. Cancer Chemother Pharmacol 1991; 27: 253–257. jects without apheresis has not been reported in detail. 4 Maruyama K, Tsuji K, Tanaka R et al. Characterization of peripheral blood progenitor cells mobilized by nartograstim The mechanism(s) of the delayed-onset thrombocytop- (N-terminal replaced granulocyte colony-stimulating factor) in enia is unclear. However, there are two speculative mech- normal volunteers. Bone Marrow Transplant 1998; 22: 313– anisms. First, thrombocytopenia caused by rhG-CSF 320. administration is most likely due to redirection of hemato- 5 Grigg AP, Roberts AW, Raunow H et al. Optimizing dose poiesis in the direction of neutrophil production. The and scheduling of filgrastim (granulocyte colony-stimulating megakaryocyte series may be suppressed by the excessively factor) for mobilization and collection of peripheral blood pro- stimulated granulocyte series. Second, several recent genitor cells in normal volunteers. Blood 1995; 86: 4437– reports suggested that rhG-CSF administration stimulated 4445. up to a hundred-fold increase in circulating hematopoietic 6Ho¨glund M, Smedmyr B, Simonsson B et al. Dose-dependent progenitor cells,30–32 resulting in the development of extra- mobilisation of haematopoietic progenitor cells in healthy volunteers receiving glycosylated rHug-CSF. Bone Marrow medullary hematopoiesis in reticuloendothelial organs such Transplant 1996; 18: 19–27. as the spleen and liver. Thrombocytopenia, splenic enlarge- 7 Tanaka R, Matsudaira T, Aizawa J et al. Characterization of ment, splenomegaly, and splenic rupture have been reported peripheral blood progenitor cells (PBPC) mobilized by filgra- in patients receiving repeated-doses of rhG-CSF.33,34 There- stim (rHuG-CSF) in normal volunteers: dose–effect relation- fore, delayed thrombocytopenia observed in the present ship for filgrastim with the character of mobilized PBPC. Br study may also be due to the splenic extramedullary hema- J Haematol 1996; 92: 795–803. topoiesis. As shown in Figure 5 and Table 1, the time of 8 Harada M, Nagafuji K, Fujisaki T et al. G-CSF-induced mobi- thrombocytopenia corresponded with the time of transamin- lization of peripheral blood stem cells from healthy adults for ase increase, which was possibly of liver origin, an another allogeneic transplantation. J Hematother 1996; 5: 63–71. reticuloendothelial organ. Increased serum levels of LDH 9 Arbona C, Prosper F, Benet I et al. Comparison between once a day vs twice a day G-CSF for mobilization of peripheral and ALP were observed prior to thrombocytopenia and blood progenitor cells (PBPC)in normal donors for allogeneic transaminase increase, but just following bone pain PBPC transplantation. Bone Marrow Transplant 1998; 22: (Figure 6), probably due to an increased turnover of gra- 39–45. nulocytes mobilized by rhG-CSF in healthy volunteers. 10 Majolino I, Cavallaro AM, Bacigalupo A et al. Mobilization These changes are all consistent with the marrow pool and collection of PBSC in healthy donors: a retrospective expansion of myelogenous progenitor cells. analysis of the Italian bone marrow transplantation group In summary, these preliminary results for the purpose of (GITMO). Haematologica 1997; 82: 47–52. PBSC transplantation suggest that repeated doses of rhG- 11 Cleaver SA, Goldman JM. Use of G-CSF to mobilise PBPC CSF induce mobilization of PBSC in a dose-dependent in normal healthy donors – an international survey. Bone Mar- manner and the predose baseline value of PBSC may pre- row Transplant 1998; 21 (Suppl. 3): S29–S31. 12 Ordemann R, Ho¨lig K, Wagner K et al. Acceptance and feasi- dict the degree of post-dose maximal mobilization. The bility of peripheral stem cell mobilisation compared to bone drug treatment may cause delayed-onset moderate throm- marrow collection from healthy unrelated donors. Bone Mar- bocytopenia and transaminase increase after administration, row Transplant 1998; 21 (Suppl. 3): S25–S28. and drug clearance changes in a complex manner. These 13 To LB, Haylock DN, Simmons PJ et al. The biology and clini- should be kept in mind during repeated dosing. cal uses of blood stem cells. Blood 1997; 89: 2233–2253.

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